Anthrax is a well-known infectious disease caused by a Gram-positive bacterium, purified Bacillus anthracis (B. anthracis). Among the three types of anthrax infection (cutaneous, gastrointestinal, and inhalation), cutaneous anthrax is the most common and is relatively easily treatable with various antibiotics (6). [It is noted that this numeral reference, and others that similarly follow, references a correspondingly numbered citation in the Literature Cited section, infra.] The other two types of anthrax are rare, but usually fatal even with aggressive anti-microbial therapy. For example, only about one fifth of those who contracted inhalation anthrax recovered in a reported outbreak that occurred in the former Soviet Union town of Sverdlovsk (28). Inhalation anthrax generally occurs after an incubation time of one to six days (10). After the incubation period, a nonspecific flu-like illness ensues for one to three days followed by a brief intervening period of improvement. Unfortunately, rapid deterioration follows and death is universal in untreated cases. Death may occur in as many as 95 percent of treated cases if therapy is not begun within 48 hours from the onset of initial symptoms (5).
Although well characterized as a disease, only in last twenty years has the molecular basis of B. anthracis virulence related to the disease been understood. The virulence of B. anthracis for animals and humans depends on the production of two types of virulence factors: the gamma-linked poly-D-glutamic acid capsule (27) and the three-component protein exotoxin that is termed anthrax toxin (23, 45). The genes related to virulence are located in two mega-plasmids: pXO1 and pXO2. The genes involved in toxin production are located in the 185 kilobase pair (kbp) pXO1 plasmid (29, 42, 46) and the genes required for capsule production are located in the 95 kbp pXO2 plasmid (13, 31, 42, 49). A B. anthracis strain that contains both plasmids is generally regarded as a virulent strain. There are many B. anthracis strains known in the art that lack virulence due to the absence of either or both of these plasmids. For example, strains lacking the pXO1 plasmid are avirulent (17, 48). Known strains without the pXO2 plasmid are at least 105-fold less virulent than wild-type strains containing both plasmids (17, 52).
The major virulence factor, anthrax toxin, is composed of three proteins: protective antigen (PA, 83 kilo Dalton, kDa), edema factor (EF, 89 kDa), and lethal factor (LF, 90 kDa). The toxin components act in the binary combinations of PA+EF (edema toxin), and PA+LF (lethal toxin). PA is a cell receptor-binding protein and delivers the other two proteins (EF and LF) into the cytosol of infected cells (3, 39). EF is a calmodulin-dependent adenylate cyclase that disables phagocytes and other cells (22).
Increased cellular levels of cyclic adenosine monophosphate (cAMP) upset water homeostasis and are believed to be responsible for the massive edema seen in cutaneous anthrax infections. Edema toxin inhibits neutrophil function in vitro (30) and neutrophil function is impaired in patients with cutaneous anthrax infection (1). Lethal toxin can be fatal to animals and certain cultured cells due to the LF action. LF is a zinc metalloprotease (14, 15, 21) that inactivates mitogen-activated protein kinase kinase (8, 38, 50). The genes encoding PA (pagA), EF (cya), and LF (lef) have all been identified and cloned (43, 47, 51, 53). The crystal structures of all three proteins have also been identified (7, 24, 39).
The most effective known method for preventing anthrax is vaccination. The current and only FDA-approved anthrax vaccine in the United States (produced by BioPort Corporation of Lansing, Mich. under the trademark BIOTHRAX) is produced from a sterile culture filtrate from an avirulent B. anthracis V770-NP1-R strain. The vaccine primarily consists of PA, and aluminum hydroxide is used as an adjuvant (10). The vaccine was developed during the 1950s and 1960s (2, 54) and is licensed by the FDA to BioPort Corporation (and was formerly licensed to the Michigan Biologic Product Institute) since 1970. The vaccine is safe, showing less than 0.06% systemic reactions (11). The ability of the vaccine to elicit an immune response in humans is well-documented (20, 40, 41). The prior art BIOTHRAX vaccine is currently licensed for six doses over 18 months followed by annual boosts. Nevertheless, data indicate that all six doses may not be necessary to elicit full immune responses (41).
Although the prior art vaccine is effective and safe, there exists in the art a desire to develop new immunogenic compositions for preparing a vaccine which protects a subject against lethal a infection B. anthracis using current recombinant technologies. Such technologies could increase characterization of the vaccine protein components and allow use of new types of adjuvants that could elicit enhanced or more diverse immune responses.
Attempts in the art are known to develop new compositions to prepare a vaccine against anthrax using more current technologies. See for example, U.S. Patent Applications 2002/0051791, 2002/0197272, 2003/0003109, to Galloway et al. Unfortunately, Galloway et al. compositions to prepare vaccines are based on DNA vaccine technology and shows very low level of immune responses (both IgG1 and IgG2a) even after 4 vaccinations (3 DNA vaccinations plus one protein vaccination).